表面标志物Cripto-1表达对食管癌干细胞生长的作用机制研究
Active mechanism of the surface marker CR-1 expression on the esophageal cancer stem cell growth
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摘要: 目的：探讨表面标志物Cripto-1（CR-1）的表达对食管癌干细胞生长的作用机制。方法：自食管癌Eca109细胞中分选Eca109干细胞，提取正常食管上皮细胞HEEC和Eca109细胞、Eca109干细胞总蛋白，采用Western-blot法检测CR-1表达水平；构建CR-1基因沉默载体CNE2/CR-1-及过表达载体CNE1/CR-1+，利用脂质体转染法将CNE2/CR-1-和CNE1/CR-1+分别转染进Eca109干细胞中，并设对照组，采用RT-PCR法检测CR-1表达；MTT法检测对照组、CNE2/CR-1-组、CNE1/CR-1+组Eca109干细胞增殖情况；流式细胞仪检测各组Eca109干细胞凋亡情况；Western-blot法检测各组细胞Akt、p-Akt蛋白表达情况。结果：Eca109细胞及Eca109干细胞中CR-1表达均明显高于HEEC（P<0.01）。RT-PCR结果显示，CNE2/CR-1-组CR-1表达水平明显低于对照组，CNE1/CR-1+组明显高于对照组（P<0.01）。MTT结果显示，CNE2/CR-1-组细胞增殖明显低于对照组，CNE1/CR-1+组明显高于对照组（P<0.01）。流式细胞仪检测显示，CNE2/CR-1-组细胞凋亡率明显高于对照组，CNE1/CR-1+组细胞凋亡率明显低于对照组（P<0.01）。Western-blot结果显示，CNE2/CR-1-组p-Akt蛋白表达明显低于对照组，CNE1/CR-1+组p-Akt蛋白表达明显高于对照组（P<0.01）；而Akt蛋白表达未出现明显变化（P>0.05）。结论：CR-1作为潜在的食管癌干细胞标记物，可能通过PI3K/Akt信号通路调控食管癌干细胞的增殖与凋亡。Abstract: Objective:To investigate the active mechanism of the surface marker Cripto-1(CR-1) expression on esophageal cancer stem cell growth.Methods:Eca109 stem cells was isolated from Eca109 cells.The total proteins of HEEC,Eca109 cells and Eca109 stem cells were extracted,and the expression level of CR-1 was detected using Western-blot.The silencing CNE2/CR1- vector targeting CR-1 gene and overexpression vector CNE1/CR-1+ were constructed,and transfected into the Eca109 stem cells using lipofectamine.The control group was set.The transfection efficiency was examined using RT-PCR.The proliferation of Eca109 stem cells in control group,CNE2/CR-1- and CNE1/CR-1+ were detected using MTT.the prognosis of Eca109 stem cells in each group was examined using flow cytometer.The protein expression levels of Akt and p-Akt in each group were detected using Western-blot.Results:The expression levels of CR-1 in Eca109 cells and Eca109 stem cells were significantly higher than that in HEEC cells(P<0.01).RT-PCR results showed that the expression level of CR-1 in CNE2/CR-1-group was significantly lower than that in control group,and the expression level of CR-1 in CNE1/CR-1+ group was significantly higher than that in control group(P<0.01).MTT assay results showed that the cell proliferation in CNE2/CR-1- group was significantly lower than that in control group,and which in CNE1/CR-1+ group was significantly higher than that in control group(P<0.01).Flow cytometry results showed that the apoptosis rate of cells in CNE2/CR-1- group was significantly higher than that in control group,which in CNE1/CR-1+ group was significantly lower than that in control group(P<0.01).Western-blot results showed that the expression level of p-Akt protein in CNE2/CR-1- was significantly lower than that in control group,and the expression level of p-Akt protein in CNE1/CR-1+ group was significantly higher than that tin control group(P<0.01).The expression change of Akt protein was not obvious(P>0.05).Conclusions:The CR-1,an esophageal cancer stem cell marker,may regulate the proliferation and apoptosis of esophageal stem cell by PI3K/Akt signaling pathway.
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Key words:
- esophageal cancer /
- stem cell marker /
- proliferation /
- apoptosis
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